Natural Killer Cell Subsets and IL-2, IL-15, and IL-18 Genes Expressions in Chronic Kidney Allograft Dysfunction and Graft Function in Kidney Allograft Recipients.

BACKGROUND
While acute rejection and early graft loss rates have decreased substantially over the past four decades, progressive chronic allograft dysfunction (CAD) still remains a common cause of late graft loss in kidney transplant recipients.


OBJECTIVE
This study was conducted to investigate the percentage of natural killer (NK) cell subsets and IL-2, 15 and 18 genes expression in two groups of CAD and well-function graft (WFG) recipients.


METHODS
30 renal allograft recipients with biopsy-proven interstitial fibrosis/tubular atrophy (IF/TA) and impaired renal function, and 30 sex- and age-matched WFG patients were enrolled in this study. The percentage of NK cell subsets including NK CD56bright and NK CD56dim cells were determined by flowcytometry; IL-2, IL-15, and IL-18 genes expressions were assessed by real-time PCR.


RESULTS
Compared to WFG patients, there was a significant (p<0.05) increase in the percentage of NK CD56bright cells in CAD patients. However, the difference in percentage of NK CD56dim cells or CD56dim/CD56bright ratio between the studied groups was not significant. In addition, IL-2, 15 and 18 genes expressions were almost similar in CAD and WFG patients.


CONCLUSION
We found higher percentages of NK CD56bright subset in kidney transplant recipients with CAD without considerable changes in related cytokines' gene expression, suggesting a possible defect of NK cells maturation in these patients.


INTRODUCTION
K idney allograft transplantation is the treatment of choice for end-stage renal diseases. Both antigen-dependent (e.g., HLA mismatch) and antigen-independent risk factors (e.g., ischemia reperfusion injury [IRI]) can result in inflammation, tissue damage, and initiation of graft failure [1]. Many studies in the context of solid organ transplantation suggest a considerable role for innate immunity, particularly natural killer (NK) cells and related cytokines, in allograft damages. However, the exact mechanisms have not yet been fully understood [2].
NK cells phenotypically express CD16 and CD56 in the absence of TCR/CD3 complex.
Considering the amount of CD56 expression, NK cells are classified as CD56 bright and CD-56 dim cells, but the expression of CD56 is not steady and changes during different stages of maturation. Mature NK cells express NKG2D, low levels of CD56, and high levels of surface CD16. While around 90% of human peripheral blood NK cells are CD56 dim and regarded as the classical cytotoxic NK cell subset, the remaining 10%, the so-called CD56 bright subset, display low cytotoxic capacity and produce high levels of IFNγ and TNFα [3].
NKCD56 dim cells produce cytolytic molecules, like perforin, granzyme, and granulyzin, to eliminate unfamiliar allograft cells [4]. But, recent studies have shown presence of regulatory NK cell subsets in stable grafts, indicating a unique role for NK cells in maintenance of homeostasis, introducing them as active participants in rejection or acceptance of transplanted organs [5]. NK cells effect on dendritic cells maturation and activation has been investigated in some studies [6,7]. For instance, it has been shown that early events following kidney transplantation involving NK-DC interaction via KIR-HLA-C immune synapse and NK cells cytolytic activity against donor-derived APCs influence the transplant outcome [8].
Tissue biopsy is the gold-standard diagnostic test for determining chronic allograft damages, but its invasiveness and consequential side effects limit its feasibility in clinic. Therefore, there is a growing tendency to replace biopsy with a battery of tests for biomarkers in peripheral blood and urine. A combination of NK cells percentages and ratio might be an eligible candidate for the new test [9]. In addition, manipulating the number and activity of NK cell subsets to find new immunomodulatory strategies seems to be helpful.
In this study we investigated NK CD3 neg CD-16 dim CD56 bright and CD3 neg CD16 bright CD-56 dim subsets among two groups of patientsa group with chronic allograft dysfunction (CAD) with biopsy-proven interstitial fibrosis/ tubular atrophy (IF/TA), and another group of recipients with stable graft function (WFG) in order to find any correlations between clinical status and NK cells counts and ratio in their peripheral blood.

Patients
Sixty adult renal transplant recipients, who had received kidney allograft between six months and five years prior to our study, were enrolled in this study. They included 30 patients with biopsy-proven CAD and 30 patients with clinically well-function graft (WFG).
The patients with CAD (n=30) had had a progressive deterioration in their allograft function with 15% or more irreversible rise in creatinine within 1-3 months and proteinuria >1 g/24 h [9]. Consequently, they were biopsied where the pathologic lesions (IF/TA) were reported in all of them [10,11].
Those with WFG (n=30) were selected according to their sex, age and time post-transplantation in order to be pair-matched with CAD patients. They were normal in clinical examination and their serum creatinine levels were ≤1 mg/dL, they had proteinuria <0.5 g/24 h and their Cockcroft creatinine clearance was >80 mL/min [9]. They had no registered history of acute rejection episodes. Since these patients presented no deterioration in their graft function, and protocol biopsies are not routine in our centers, no biopsy was taken for this group. None of the enrolled patients had diagnosed with infectious diseases at the sampling time. The protocol conformed to the ethical guidelines of the 1975 Helsinki Declaration and was approved by the Ethics Committee of Tehran University of Medical Sciences. All patients gave written informed consent prior to the inclusion in our study.

Flowcytometry
For staining of CD3 neg CD16 dim CD56 bright and CD3 neg CD16 bright CD56 dim NK cells, we used FITC anti-human CD16, APC anti-human CD56, and PE anti-human CD3 (eBioscience, San Diego, USA). FITC conjugate mouse IgG1, mouse IgG1 isotype control PE, and mouse IgG1 isotype control APC (eBioscience, San Diego, USA) were also used as isotype negative controls. Flowcytometry was performed by FACSCalibur (BD FacsCalibur Becton Dickinson, USA) instrument and data were analyzed by CellQuest Pro software.

RNA Isolation and Real-Time PCR
RNAs were isolated from PBMC using high pure RNA isolation kit (Roche Diagnostics, Mannheim, Germany) according to manufacturer's instruction. RNA quality was assessed by NanoDrop1000 spectrophotometer (Thermo Scientific, USA) and samples with A 260 /A 280 ratio of 1.8-2.2, and A 260 /A 230 ratio of 2-2.2 were considered "acceptable." RNA samples reverse transcription to cDNA was performed by transcriptor first strand cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany). cDNA quality was also evaluated by NanoDrop1000 spectrophotometer (Thermo Scientific, USA) and samples with A 260 / A 280 ratio of 1.7-2 were stored at -70 °C until use.
Gene expression assay was performed by realtime PCR using TaqManprobs and specific primers supplied by ABI (ABI, Applied Bio-systemStepOnePlus). The endogenous control was housekeeping gene, β-actin. Relative gene expression was calculated by standard curve method using ∆∆C t value for each amplified patient sample and cDNA from a healthy control. Data were analyzed by Applied Biosys-temStepOne software v2.1 [12].

Statistical Analysis
Data were presented as mean±SD. Comparison between groups was performed by non-parametric Kruskal-Wallis and one-way ANOVA tests. A p value <0.05 was considered statistically significant.

RESULTS
Demographic and basic characteristics of studied groups are shown in Table 1. Regarding the percentage of NK cell subsets in lymphocyte population, CAD group presenting higher percentages of NK CD56 bright cells compared to the WFG recipients (p<0.05). However, no difference was observed in NK CD56 dim cells frequency or CD56 dim /CD56 bright ratio between the two groups ( Table 2).   Table  3).
Comparison of IL-2, IL-15, and IL-18 genes expression between the studied groups showed no significant variation between patients with CAD and WFG (Table 4).
According to the inclusion criteria set, none of patients had infectious diseases at time of sampling. However, a number of patients had past medical history of CMV positivity (IgM and IgG antibody to CMV). Therefore, the correlation between CMV positivity history and NK cells subsets frequency was evaluated in these patients. NK CD56 dim cells level was significantly higher among WFG patients with history of CMV positivity compared with CMV-negative patients (p=0.025) ( Table 5).
No significant difference was observed in NK CD56 dim and NK CD56 bright cell percentages among renal allograft recipients receiving different immunosuppressive regimes including cyclosporine (Sandimmune), tacrolimus, and rapamycin.

DISCUSSION
NK cells, an important part of innate immunity, play a critical role in host defense against intracellular microorganisms and tumor cells by exerting cytotoxic activity and producing cytokines like INF-γ [13]. The NK cells controversial role in transplantation has been also considered in order to find a way to modulate their various activities for reaching better allograft survival [14].
Some of well-known disadvantages of presence of NK cells in allograft are killing donor cells according to the "missing self " theory [1], activating T cells via INF-γ secretion and OX40-OX40L interaction [15,16], contribution to antibody-mediated injuries by antibody-dependent cytotoxicity (ADCC) mechanism [17], and predisposing endothelial damage because of MICA recognition by NK-G2D on NK cells, which results in enhanced cytotoxicity [18,19].
On the other hand, there are studies in favor of NK cells positive effects on allograft. For instance, Laffont, et al, showed that NK cells    [21]. Recent studies also indicate involvement of these cells in CD4 + and CD8 + T cell regulation [22,23]. The role of NK cells in inducing chronic allograft vasculopathy has also been shown in a study [24]. Furthermore, new findings suggest therapeutic modalities that interfere with NK cells function in order to selectively modulate their cytolytic activity against donor APCs and help inducing tolerance, especially in highly immunogenic transplants [6].
In the present study, we investigated NK cell subsets in two sex-and age-matched groups of renal allograft recipients with different transplantation outcomes within similar posttransplantation intervals. Regarding the two major peripheral NK cell subsets, the CD-56 bright NK cell subset was found to be significantly increased in CAD patients in comparison with WFG patients. However, there was a similar percentage of NK CD56 dim in studied groups. Although this finding can be partially explained by the effect of immunosuppressive drugs on development of NK cells, other probable underlying causes have to be investigated.
We also analyzed NK CD56 dim /NK CD56 bright cells ratio, and found that it was also similar in WFG and CAD recipients. We could not find any association between advanced and mild pathological grades of IF/TA and NK subsets percentage.
There are evidence for the effect of various cytokines on NK cell maturation and activation; for example, IL-2 helps NK CD bright cells to produce INF-γ [25], and IL-1, IL-2, IL-12, IL-15 and IL-18 contribute to NK CD dim cells cytolytic activity [26,27]. IL-2 and IL-12 cytokines genes polymorphism have been investigated in various immunologic disorders [28,29]. Therefore, we evaluated gene expression of a cytokine profile to find any differences between WFG recipients and patients with CAD. Nevertheless, IL-2, IL-15, and IL-18 genes represented similar ranges of expression in these groups.
There are also studies indicating inhibitory effect of cyclosporine-A on NK cells global activation [30]. Some researchers showed that cyclosporine-A reduces the number of NK CD56 dim cells and causes a significant increase in NK CD56 bright subset frequency [31,32]. However, considering prior evidence about this issue, we did not find any significant correlation between immunosuppressive regimens and peripheral NK cell repertoire of kidney transplant recipients, probably because of low study power.
In conclusion, the present study demonstrated a significant increase in NK CD56 bright cells among CAD patients with biopsy-proven IF/ TA lesions without considerable alteration in related cytokines gene expression in comparison with WFG recipients, suggesting a possible defect in NK cell maturation in these patients. Nonetheless, larger detailed studies are required to further assess the function and relevance of NK cells in renal transplantation long-term outcomes.